PROJECT SUMMARY Glutamine and other amino acids are important nutrients that support the metabolic and biosynthetic reactions necessary to sustain tumor growth. Elevated consumption rates by tumor cells can lead to a tumor ecosystem that is depleted of nutrients; however, tumors have the capacity to evolve metabolic adaptations that allow them to circumvent such nutrient stress. One such adaptation is the stimulation of macropinocytosis, an endocytic uptake mechanism that functions as an amino acid supply route in Ras-transformed cells. By enabling the uptake of extracellular protein and targeting it for lysosomal degradation, the macropinocytosis pathway supplies tumor cells with amino acids, allowing tumors to circumvent nutrient depletion and survive nutrient stress. Hence, the blockade of macropinocytosis represents a novel intervention strategy to starve tumor cells of nutrients; however, the signaling networks that control and regulate macropinocytosis in tumors are insufficiently understood. In this proposal, we will investigate the molecular mechanisms that drive macropinocytic induction in response to nutrient stress. Our preliminary data provide the first lines of evidence indicating that nutrient stress elicited by glutamine depletion has the ability to modulate macropinocytosis in a subset of Ras-transformed pancreatic cancer cells. This is important because pancreatic cancer has been recently recognized as a recalcitrant cancer that is in urgent need of new therapeutic strategies that offer an improvement in clinical outcome. We have attributed nutrient stress-induced macropinocytosis to the activation of the EGFR pathway and, importantly, we have implicated p53 as critical to controlling this EGFR-dependent uptake. In this proposal, we anticipate deciphering how this inducible form of macropinocytosis relies on EGFR and how this process is orchestrated by nutrient stress signals emanating from p53. Therefore, we will test the hypothesis that pancreatic tumors cope with nutrient stress by boosting their macropinocytic capacity, a process that is driven by EGFR signaling and p53. We will: (1) Determine the role of the EGFR signaling pathway in glutamine depletion-induced macropinocytosis; (2) Examine the functional interplay between oncogenic and wild-type Ras during nutrient stress; and (3) Decipher the function of p53 as a nutrient stress sensor that activates EGFR-dependent macropinocytosis. These aims will be investigated utilizing a combination of in vitro cell-based assays, human tumor interrogation, and mouse models of pancreatic cancer. In summary, our work could establish macropinocytosis as a critical metabolic adaptation that supports tumor cell fitness during pancreatic cancer progression and set the stage for new drug discovery efforts that exploit the nutrient dependencies of tumors.